Method for creating a cell growth surface on a polymeric substrate

ABSTRACT

A method, apparatus and product for producing an advantaged cell growth surface. According to the present invention, a stream of plasma is comprised of activated gaseous species generated by a microwave source. This stream is directed at the surface of a polymer substrate in a controlled fashion such that the surface is imparted with attributes for cell adhesion far superior to that of untreated polymer or polymer treated by other known methods.

FIELD OF INVENTION

The present invention relates generally to the field of cell growthlaboratory ware and more specifically to a method of treating thesurface of a polymer in order to create a product that facilitates cellgrowth. An apparatus for performing the surface treatment is alsoprovided by the present invention.

BACKGROUND

The cultivation of living cells is a key component in, among otherthings, the drug discovery process. Many devices are sold for purposesof cell culture including roller bottles, flasks, dishes, well plates,cell harvesting units, etc. Typically these items of laboratory ware aremolded from polymers having a sufficient mechanical stability andstrength to create the necessary substrate surface for cell attachmentand growth.

Generally, cell growth containers or substrates need to be surfacetreated after molding in order to make the surface hydrophilic and toenhance the likelihood for effective cell attachment. Surface treatmentmay take the form of a surface coating, but typically involves the useof directed energy at the substrate surface with the intention ofgenerating chemical groups on the polymer surface. These chemical groupswill have a general affinity for water or otherwise exhibit sufficientpolarity to permit stable adsorption to another polar group. Thesefunctional groups lead to hydrophilicity and or an increase in surfaceoxygen and are properties recognized to enhance cell growth. Suchchemical groups include groups such as amines, amides, carbonyls,caboxylates, esters, hydroxyls , sulfhydryls and the like. Examples ofdirected energy include atmospheric corona discharge, radio frequency(RF) vacuum plasma treatment, and DC glow discharge. These polymersurface treatment methods have displayed varying degrees of success andtheir effects tend to decay overtime.

In the case of plasma treatment, plasmas are created when a sufficientamount of energy, is added to gaseous atoms and/or molecules, causingionization and subsequently generating free electrons, photons, freeradicals, and ionic species. The excitation energy supplied to a gas toform a cold plasma can originate from electrical discharges, directcurrents, low frequencies, radio frequencies, microwaves or other formsof electromagnetic radiation. Plasma treatments are common for surfacemodification in the microelectronic and semiconductor industries. Asmentioned, atmospheric corona and RF plasma treatment are commonly usedfor polymeric surface activation for cell growth substrates as well asmedical implants.

Current standard practices for growing adherent cells in cell cultureinvolves the use of defined chemical media to which is added up to 10%volume bovine or other animal serum. The added serum provides additionalnutrients and/or growth promoters. In addition serum proteins promotecell adhesion by coating the treated plastic surface with a biolayermatrix to which cells can better adhere. The addition of serum istypically required to support the normal growth of the majority of celllines. While advantageous for cell growth, serum can have adverseeffects by intruding sources of infection or abnormally inducingexpression of unwanted genes exposed to serum.

SUMMARY OF INVENTION

According to the present invention, a stream of plasma is comprised ofactivated gaseous species generated by a microwave source. This streamis directed at the surface of a polymer substrate in a controlledfashion such that the surface is imparted with attributes for celladhesion far superior to that of untreated polymer or polymer treated byother known methods. The treatment apparatus contains a microwavegenerator and gas line feeding into a plasma mixing chamber. The plasmamixing chamber is connected to a dual chambered treatment chamber,comprising an inner chamber and an outer chamber. The outer chamberconnects directly to the plasma mixing chamber and has a vacuum lineoutlet in order to create a plasma flow. The inner chamber is containedwithin the outer chamber and contains a baffle that directs the plasmaflow directly onto the polymer surface which is to be treated. The partthat has been subjected to the directed plasma stream is imparted withuniform surface characteristics that enable extraordinary levels of cellattachment even under reduced serum conditions. It will be obvious toone skilled in the art that this surface may also be advantageous inprotein binding assays.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic drawing of the microwave plasma treatmentapparatus of the present invention.

FIG. 1A is a three-dimensional view of the inner chamber one embodimentof the present invention.

FIG. 2 is a schematic drawing of an embodiment of the microwave plasmatreatment apparatus of the present invention.

FIG. 3 is a schematic drawing of an embodiment of the microwave plasmatreatment apparatus of the present invention.

FIG. 4 is an AFM micrograph of a surface treated in accordance with thepresent invention, after being exposed to water.

FIG. 5 is an AFM micrograph of a surface treated in accordance with thepresent invention, exposed only to air.

FIG. 6 is an AFM micrograph of a surface treated in accordance with aprior art radio frequency plasma method.

FIG. 7 is a graphical representation comparing the contact angle ofsubstrate surfaces treated in accordance with the present invention andsurfaces treated with a prior art radio frequency plasma method.

FIG. 8 is a graphical representation of a comparative cell growth studyperformed with injection molded polystyrene flasks from sampling ofmanufacturers and that have been treated with a variety of differentmethods, and comparing the microwave plasma method of the presentinvention.

DETAILED DESCRIPTION

With reference to FIG. 1, a basic construction of the microwave plasmastream apparatus for carrying out the method of the present invention isprovided. A 2.45 GHz microwave generator 10 (MKS Astex, Wilmington,Mass.) serves as the energy source of this apparatus. The equipmentpreferably includes a generator, circulator, dummy load, tuner, andapplicator. A gas line 12 connects to a gas source and delivers theprocess gas, which when sufficiently energized creates a continuousstream of activated or ionized gas. Suitable plasma gases include argon,nitrogen, oxygen, nitrous oxide, ammonia, carbon dioxide, helium,hydrogen, air and other gases known to those of skill in the art toreadily be activated or ionized. A plasma chamber 14 serves as amanifold for the reaction between gas and microwave energy, and is influid communication with both the gas line 12, via a valve 13, as wellas the microwave generator 10. A conduit 16 connects the plasma chamberwith a treatment chamber 18 through an aperture 20. Within the first orouter treatment chamber 18, a second or inner treatment chamber 22 islocated. The inner chamber has a frusto-conical baffle section whichserves to contain the plasma flow and direct it onto a part that isplaced at its base. In this embodiment, the inner chamber shares acommon base 25 with the outer chamber. Further, it is preferred that theinner treatment chamber have a top neck portion which roughly matchesthe aperture 20 in cross sectional area. However, it is preferred thatthe neck of the inner treatment chamber not connect directly to theaperture. The approximate 1-6 inch gap between the aperture and the neckof the second treatment chamber enable the plasma to flow out of theouter treatment chamber through a valved vacuum line 24. A pneumaticelevating system 29 may be employed to move the base portion 25 awayfrom the treatment chamber in order to remove treated parts and placenew parts into the inner chamber in an automated fashion. Preferably,the plasma mixing chamber 14 is of quartz construction. The conduit 16and outer treatment chamber, may be made from conductive ornonconductive materials, especially quartz ,aluminum or stainless steel.The inner treatment chamber is preferably made from a nonconductivematerial, and most preferably, quartz.

In operation, the apparatus of FIG. 1 performs as follows: A moldedpolymer part to be treated is located within the inner chamber 22. Forpurposes of illustration, a multiwell plate 26 has been placed on thebase 25, but the inner and outer chamber may be shaped, dimensioned andconfigured to accommodate any of a variety of polymer parts. A vacuumseal is created between the base 25 and the sidewalls 27 of the outerchamber. To enable continuous flow, vacuum pumping is maintained throughthe process. The valves 13, 23 are opened and the process gas is allowedto flow into the plasma chamber 14, through the aperture 20 and into thedual chambered treatment area. The gas flows at a pressure preferablybetween 100 and 2,000 millitorr, and more preferably between 200 and 300millitorr. The gas preferably set to flow at a rate of 100 to 5,000cc/min, and more preferably between 400 and 600 cc/min. While theprocess may run at any range of temperatures, it preferably runs between40 and 150 degrees Fahrenheit and more preferably at room temperature,or approximately 72 degrees Fahrenheit. The microwave generator isengaged to create an output of between 300 and 10,000 watts, andpreferably between 300 and 3,000 watts. The microwave energy enteringthe plasma chamber 14 interacts with the gas entering the plasma chamberresulting in activation of the gas thereby creating the resultantplasma. Due to the constant flow characteristics of the assembly, theplasma is directed through the conduit 16, through the aperture 20, andinto the treatment chamber. The stream or jet created by the plasma flowthrough the conduit and aperture is directed into the outer treatmentchamber 18, subsequently into inner treatment chamber 22, and onto thepolymer part 26 placed at the base 25 of the chamber. Flow out of boththe inner chamber 22 and outer chamber 18 is assured due to the vacuumline 24, which serves to evacuate the dual chambered treatment area. Itshould be noted that due to the inner treatment chamber 22, the plasmastream is directed onto the part as opposed to directly toward theoutlet valve 23, thereby enabling the part 26 to have optimal contactwith the stream. The inner treatment chamber 22 should be entirelyenclosed and sealed from the outer chamber 18, but for the opening atthe neck. A three-quarters view of the inner chamber is shown in FIG.1A. A neck portion 4 and a funnel portion 6 make up the frusto-conicaltop portion. In this embodiment, the base 8 is rectangular in shape soas to receive a well plate.

The plasma is energized for between 1 second and 5 minutes and morepreferably for between 5 and 20 seconds. Once treatment is complete, themicrowave energy is ceased, valves are closed, an atmospheric vent valve32 is opened to introduce nitrogen or dry air to the system and in orderto return all the chambers to atmospheric pressure. After normalizationof pressure, the part is removed by operating the pneumatic elevatingsystem 29. Optimally, a computer control system performs the stepsoutlined above in an automated fashion. After removal, the part ispreferably given a standard sterilization treatment by exposure to gammaradiation.

FIG. 2 is a schematic representation of another embodiment of thepresent invention. In this embodiment, it is the part to be treated thatacts as the inner treatment chamber. As is the previously describedembodiment the apparatus has a gas inlet 12 and a microwave generator 10in communication with a plasma chamber 14. A plasma stream is created byflow from line 24 which is attached to a vacuum pump. The plasma streamis created by plasma moving though the conduit 16 and aperture 20 andinto the outer treatment chamber 18. However, in this case, the part tobe treated, a roller bottle 30 serves as the ‘inner chamber’. The bottle30 is placed close to the aperture, approximately 1-6 inches away, suchthat the plasma stream will be directed into the bottle. The plasmastream is directed through the neck of the bottle and contacts all innersurfaces of the bottle including bottom and sidewalls. Again, anatmospheric vent 32 connecting with the outer treatment chamber isemployed for pressure equalization in removing the part. As in thepreviously described embodiment, a pneumatic elevating system 33 may beemployed for removal of the part as well as to bring neck portion of thepart 30 into close proximity with the aperture 20 at the top of theouter treatment chamber 18.

FIG. 3 is a schematic representation of still another embodiment of thepresent invention. As in the previous embodiment, it is the part itselfthat serves as the inner treatment chamber. The part displayed in thisembodiment is a flask. The apparatus has a gas inlet 12 and a microwavegenerator 10 in communication with a plasma chamber 14. A plasma streamis created by flow from line 24 which is attached to a vacuum pump. Theplasma stream is created by plasma moving though the conduit andaperture 20 and into the outer treatment chamber 18. As in the previousembodiment, the part to be treated, a flask, serves as the ‘innerchamber’. The flask 40 is placed close to the aperture, preferablybetween 1 and 3 inches away, such that the plasma stream will bedirected into the flask. The plasma stream is directed through the neckof the flask and contacts all inner surfaces of the flask includingbottom and sidewalls. An atmospheric vent 32 connecting with the outertreatment chamber is employed for pressure equalization and subsequentpart removal. As in the previously described embodiments, a pneumaticactuator 42 may be employed for removal of the part 40 as well as tobring the part into close proximity with the aperture 20 at the top ofthe outer treatment chamber. In this embodiment, the conduit 16 andaperture 20 are angled to align with the angled neck of the part 40.This angling is preferable because it ensures a direct plasma streaminto the part.

The surface of the polymeric substrate to be treated can have any shape,for example it can be flat, curved or tubular. Preferably, it is a flatplanar surface. For purposes of this invention, the polymeric substratecan be biodegradable or non-biodegradable. Preferably, to be useful inboth in vivo and in vitro applications, the polymeric substrates of thepresent invention are non-toxic, biocompatible, processable, transparentfor microscopic analysis, and mechanically stable.

A large variety of polymers may be used as substrates in the articles ofthe present invention. Examples of polymers useful in the presentinvention include polyacrylates, polymethylacrylates, polycarbonates,polystyrenes, polysulphones, polyhydroxy acids, polyanhydrides,polyorthoesters, polyphosphazenes, polyphosphates, polyesters, nylons ormixtures thereof.

Examples of substrates that can be treated by the method disclosedherein include but are not limited to: flasks, dishes, flat plates, wellplates, bottles, containers, pipettes, tubes, medical devices, filterdevices, membranes, slides, and medical implants. These items aretypically formed by commonly practiced techniques such as injectionmolding, extrusion with end capping, blow molding, injection blowmolding, etc.

Although the invention is targeted for cell adhesion, attachment, andgrowth, the resultant polymer substrate surface promotes adsorption of anumber of biologically active molecules including but not limited to:peptides, proteins, carbohydrates, nucleic acid, lipids, polysaccarides,or combinations thereof, hormones, extracellular matrix molecules, celladhesion molecules, natural polymers, enzymes, antibodies, antigens,polynuceotides, growth factors, synthetic polymers, polylysine, drugsand other molecules.

Any cell type known to one of skill in the art may be attached and grownon the treated substrates of the present invention. Examples of celltypes which can be used include nerve cells, epithelial cells,mesenchymal stem cells, fibroblast cells, and other cell types.

While the mechanism for enhanced cell attachment to the substratetreated according to the present method is not fully understood, it isbelieved to stem from three general characteristics: surface morphology,chemical functionalities , and surface energy.

EXAMPLES Example 1 Surface Morphology

FIGS. 4 and 5 are AFM micrographs demonstrating surface morphology of aplasma treated surface created according to the present method. Theabove described apparatus and method were employed in order to producethe sample shown in FIGS. 4 and 5. Oxygen was used as the process gas,at a pressure of 270 millitorr, at a rate of 500 cc/min. The output fromthe microwave generator was 1500 watts and the part was exposed to theplasma stream for 20 seconds.

FIG. 4 shows the surface in water, while FIG. 5 shows the treatedsurface in air. For comparative purposes, FIG. 6 shows a surface thathas been treated by a conventional RF plasma technique (using oxygen asa process gas, at a pressure of 270 millitorr, rate of 500 cc/min, andoutput from RF of 600 watts, treated for 3 minutes) as it appears inwater. It can be noted that the surface of the microwave plasma treatedsubstrate changes significantly when exposed to water. A roughened andhigh surface area morphology develops. The surface roughness as measurein RMS (Root Mean Square) increased approximately five times with themicrowave plasma surface in liquid as compared to that in air (comparingFIG. 4 and FIG. 5). The RF surface did not undergo any significantchange when exposed to water. It is believed that this roughened surfaceexposes a greater surface anchoring area to cells for attachment.

Example 2 Contact Angle

FIG. 7 is a graphical demonstration of contact angle measurementsperformed over a two-year period on the surface of three blow-molded,treated polystyrene roller bottles. Roller bottles were treated withstandard RF plasma treatment, with microwave oxygen plasma under thesame conditions as described above, and with microwave nitrous oxideplasma, also under the same conditions as described above. All of theroller bottles used in the experiment were from the same manufacturingrun, surface treated at the same time, and subsequently gamma sterilizedat the same time and under the same dosage. As can be ascertained by thetable of FIG. 7, all three treatment methods showed an increase incontact angle over time. However, the microwave plasma treated rollerbottles show significantly lower contact angles at time zero. As aconsequence, even after over two years, the contact angle measured inthe bottles affected by the microwave plasma treatment of the presentinvention, have contact angles that are lower or equivalent to thecontact angle for the RF plasma treated substrates at time zero.

Example 3 Oxygen Content (MW Plasma v. RF Plasma)

Table 1 compares the surface chemistry of blow molded polystyrene rollerbottles treated with RF plasma, microwave oxygen plasma, microwavenitrous oxide plasma, and an untreated control. Both the microwaveplasma treatments were run with gas pressure of 270 millitorr, flow rateof 500 cc/min, output from microwave of 1500 watts and exposure time of20 seconds. The RF plasma treatment was performed under the identicalconditions described in Example 1 above. After treatment, the surfacesof the bottles were analyzed using ESCA (Electron Microscopy forChemical Analysis). This test analyzes polystyrene for percentages ofoxygen, carbon, and nitrogen species on the surface. As can be readilyobserved from the results, untreated polystyrene has approximately onehundred percent carbon species on its surface. RF plasma treatmentsignificantly increases the oxygen surface content (17.8%), and createsa slight amount of nitrogen (0.2%). The microwave treatment of thepresent method imparted a surface oxygen content significantly exceedingthat of RF plasma, (31 % higher for MW-oxygen, 37% higher for MW-N₂O)while also marginally increasing the nitrogen surface content.

TABLE 1 Sample Carbon (%) Oxygen (%) Nitrogen (%) Untreated 100 0 0 RFPlasma 82.0 17.8 0.2 MW Plasma Oxygen 76.4 23.3 0.3 MW Plasma N₂O 75.224.3 0.5

Example 4 Oxygen Content (MW Plasma v. Corona Discharge)

Table 2 compares the surface chemistry of injection molded polystyreneflasks treated with standard corona discharge techniques, microwaveoxygen plasma, microwave nitrous oxide plasma, and an untreated control.Parameters for the microwave plasma treatment were identical to thosedisclosed in Example 3 above. After treatment, the surfaces of thebottles were analyzed using ESCA. As shown in table 2, considerably moreoxygen and nitrogen content were observed respectively on the microwaveplasma treated surface when compared to the corona treated surface (32%higher for MW-oxygen, 42% higher for MW-N₂O).

TABLE 2 Sample Carbon (%) Oxygen (%) Nitrogen (%) Untreated 100 0 0Corona 78.5 21.0 0.3 MW Plasma Oxygen 72.0 27.8 0.3 MW Plasma N₂O 69.329.8 1.0

Example 5 Cell Growth

FIG. 8 is a graphical representation of a comparative cell growth studyperformed with injection molded polystyrene flasks from sampling ofmanufacturers and that have been treated with a variety of differentmethods and comparing the microwave plasma method of the presentinvention. Cell growth conditions were measured under 10% serum, 1%serum and no serum growth conditions. The cell line used was Hek-293.Cells were seeded onto all surfaces at the same time, with the sameinitial number of cells, under the same conditions. Once the first flaskwas completely filled with a confluent monolayer of cells as determinedby visual inspection, all samples were analyzed for cell count.Measurements were achieved by using a Coulter Counter™ (Beckman Coulter,Inc., Fullerton, Calif.). The sample substrates tested were, from leftto right in the graph of FIG. 8, Corning corona tissue culture treatedflask, (Corning Inc. Cat. #430641 ) microwave nitrous oxide plasmatreatment as per the disclosed method, FALCON™ tissue culture flasks(Falcon, Cat. #353111), PRIMERIA™ tissue culture flasks (Primaria, Cat.#353801), and NUNC™ tissue culture flasks (Nunc, Cat. #178891). Asdemonstrated in the graph of FIG. 8, the microwave plasma treatmentsubstrate of the present invention outperformed all commerciallyavailable cell culture substrates tested, at all three serum levels.

From the foregoing description of the various preferred embodiments, itshould be appreciated that the present invention may take many variousforms and that the present invention is to be limited only by thefollowing claims.

We claim:
 1. An apparatus for treating a polymeric substrate surfacecomprising: a) a gas inlet, a microwave energy source and a plasmamixing chamber, the plasma mixing chamber in fluid communication withboth the gas inlet and the microwave energy source; b) a dual chamberedtreatment area having an inner treatment chamber contained within anouter treatment chamber, said inner treatment chamber having an openingin fluid communication with said outer chamber; c) said plasma mixingchamber in fluid communication with said outer treatment chamber bymeans of an aperture; d) a vacuum outlet line attached to said outerchamber; and e) whereby said opening in said inner treatment chamber isaligned with said aperture, said opening being spaced from said aperturea predetermined distance.
 2. The apparatus of claim 1 wherein saidpredetermined distance is between 1 and 6 inches.
 3. The apparatus ofclaim 1 wherein said outer treatment chamber has an atmospheric ventattached thereto.
 4. The apparatus of claim 1 further comprising apolymer part within said inner chamber.
 5. The apparatus of claim 1wherein said inner chamber is a polymer part to be treated.
 6. Theapparatus of claim 1 wherein said inner treatment chamber has sidewalls,a base and a tapered neck portion defining said opening.
 7. Theapparatus of claim 1 further comprising a pneumatic elevating system forremoval of a base portion of said outer treatment chamber.
 8. Theapparatus of claim 1 further comprising a conduit attaching said plasmamixing chamber and said outer treatment chamber through said aperture.9. A method for treating the surfaces of a polymer substrate comprisingthe steps of: a) providing the apparatus of claim 1 and placing thepolymeric substrate in said treatment area; b) producing a lowtemperature plasma in said plasma mixing chamber; and c) introducingsaid plasma to said dual treatment chamber.
 10. The method of claim 9further comprising the step of evacuating said plasma from saidapparatus.
 11. The method of claim 9 further comprising the step ofplacing a polymer part into said inner treatment chamber prior to saidproducing step.
 12. The method of claim 9 wherein said inner chamber isa polymer part to be treated.
 13. The method of claim 9 wherein saidpolymer is polystyrene.